RESUMO
Cancer progression and treatment-associated cellular stress impairs therapeutic outcome by inducing resistance. Endoplasmic reticulum (ER) stress is responsible for core events. Aberrant activation of stress sensors and their downstream components to disrupt homeostasis have emerged as vital regulators of tumor progression as well as response to cancer therapy. Here, an orchestrated nanophotoinducer (ERsNP) results in specific tumor ER-homing, induces hyperthermia and mounting oxidative stress associated reactive oxygen species (ROS), and provokes intense and lethal ER stress upon near-infrared laser irradiation. The strengthened "dying" of ER stress and ROS subsequently induce apoptosis for both primary and abscopal B16F10 and GL261 tumors, and promote damage-associated molecular patterns to evoke stress-dependent immunogenic cell death effects and release "self-antigens". Thus, there is a cascade to activate maturation of dendritic cells, reprogram myeloid-derived suppressor cells to manipulate immunosuppression, and recruit cytotoxic T lymphocytes and effective antitumor response. The long-term protection against tumor recurrence is realized through cascaded combinatorial preoperative and postoperative photoimmunotherapy including the chemokine (C-C motif) receptor 2 antagonist, ERsNP upon laser irradiation, and an immune checkpoint inhibitor. The results highlight great promise of the orchestrated nanophotoinducer to exert potent immunogenic cell stress and death by reinforcing ER stress and oxidative stress to boost cancer photoimmunotherapy.
Assuntos
Neoplasias , Humanos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias/terapia , Estresse do Retículo Endoplasmático/efeitos da radiação , Estresse Oxidativo , Apoptose , Linhagem Celular TumoralRESUMO
Although endoplasmic reticulum (ER) stress is thought to be involved in various diseases such as cancer, metabolic, and inflammatory disorders, the relationship between ER stress and bone diseases, are remains unclear. Tunicamycin-treated MC3T3-E1 osteoblasts were used as the ER stress model in this study. 635 nm light-emitting diode irradiation (635 nm-IR) was carried out for 1 h before and after inducing ER stress. To investigate the effects of 635 nm-IR on ER stress-induced MC3T3-E1 osteoblasts and the underlying mechanism, western blot, reverse transcription polymerase chain reaction, alkaline phosphatase and Alizarin red staining, 2',7'-dichlorodyhydrofluorescein diacetate assay, Fluo-3AM and immunocytochemistry were performed. Pretreatment with 635 nm-IR effectively prevented intracellular reactive oxygen species production and alleviated ER stress through the pancreatic ER kinase (PERK)-eukaryotic initiation factor 2 (eIF2)-activating transcription factor 4 (ATF4)-nuclear factor-like 2 (Nrf2) signaling pathway. Hence, 635 nm-IR may serve a protective role in the treatment of ER stress-related bone diseases.
Assuntos
Estresse do Retículo Endoplasmático/efeitos da radiação , Lasers Semicondutores , Osteoblastos/efeitos da radiação , Células 3T3 , Fator 4 Ativador da Transcrição/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
Today, infertility affects 15% of couples and half of this rate is due to reproductive problems in men. Radiation-induced damage to the testicles causes sterility depending on the dose. Radiation causes endoplasmic reticulum (ER) stress and ER stress induces apoptosis. In this study, the effect of human amniotic membrane-derived mesenchymal stem cells (hAMSCs) and conditioned medium (hAMSCs-CM) on testicular damage induced by ionizing radiation is aimed to be elucidated through ER stress and apoptosis mechanisms. Six gray scrotal irradiation was used to create a testicular injury model. hAMSCs isolated and characterized with immunofluorescence and flow cytometry, while 2.5 × 105 hAMSCs were transplanted into testis and hAMSCs-CM was applied. Fertility assessment was performed. Expressions of ER stress markers GRP78, Ire1, Chop and Caspase-12, and Caspase-3 were determined. TUNEL was performed. Serum FSH, LH, and testosterone were measured. After hAMSC transplantation and administration of hAMSCs-CM, offsprings were obtained. Seminiferous tubule diameter and seminiferous epithelial height increased. The expression of GRP78, IRE1α, CHOP, Caspase-12, and Caspase-3 decreased. Percentages of tunel positive cells decreased. While FSH and LH levels decreased, testosterone increased. After irradiation, both hAMSCs transplantation and paracrine activity of hAMSCs may have a role in reducing ER stress by suppressing the UPR response. Decrease in FSH and LH and increase in testosterone level after MSCs transplantation may have contributed to the improvement of spermatogenesis. Thus, it can be said that MSCs derived from human amniotic membrane can improve ionized radiation-induced testicular damage by reducing ER stress and apoptosis.
Assuntos
Âmnio/citologia , Apoptose/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos da radiação , Infertilidade Masculina/etiologia , Infertilidade Masculina/terapia , Transplante de Células-Tronco Mesenquimais , Testículo/efeitos da radiação , Animais , Meios de Cultivo Condicionados , Feminino , Humanos , Masculino , RatosRESUMO
Breast cancer is a major threat to women's health and estrogen receptor-positive (ER+) breast cancer exhibits the highest incidence among these cancers. As the primary estrogen, estradiol strongly promotes cellular proliferation and radiotherapy, as a standard treatment, exerts an excellent therapeutic effect on ER+ breast cancer. Therefore, we herein wished to explore the mechanism(s) underlying the inhibitory effects of radiation on the proliferation of ER+ breast cancer cells. We used the ER+ breast cancer cell lines MCF7 and T47D, and their complementary tamoxifen-resistant cell lines in our study. The aforementioned cells were irradiated at different doses of X-rays with or without exogenous estradiol. CCK8 and clone-formation assays were used to detect cellular proliferation, enzyme-linked immunosorbent assay (ELISA) to determine estradiol secretion, western immunoblotting analysis and quantitative real-time PCR to evaluate the expression of proteins, and immunofluorescence to track endoplasmic reticulum stress-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays to explore the protein expression in tumors using immunohistochemistry. We found that ionizing radiation significantly reduced the phosphorylation of estrogen receptors and the secretion of estradiol by ER+ breast cancer cells. CYP19A (aromatase) is an enzyme located in the endoplasmic reticulum, which plays a critical role in estradiol synthesis (aromatization), and we further demonstrated that ionizing radiation could induce endoplasmic reticulum stress with or without exogenous estradiol supplementation, and that it downregulated the expression of CYP19A through ER-phagy. In addition, ionizing radiation also promoted lysosomal degradation of CYP19A, reduced estradiol synthesis, and inhibited the proliferation of tamoxifen-resistant ER+ breast cancer cells. We concluded that ionizing radiation downregulated the expression of CYP19A and reduced estradiol synthesis by inducing endoplasmic reticulum stress in ER+ breast cancer cells, thereby ultimately inhibiting cellular proliferation.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/radioterapia , Proliferação de Células/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos da radiação , Estradiol/biossíntese , Radiação Ionizante , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos da radiação , Animais , Aromatase/metabolismo , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Estradiol/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Resultado do Tratamento , Carga Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mammalian target of rapamycin (mTOR) is a sensor of nutrient status and plays an important role in cell growth and metabolism. Although inhibition of mTOR signaling promotes tumor cell death and several mTOR inhibitors have been used clinically, recent reports have shown that co-treatment with MHY1485, an mTOR activator, enhances the anti-cancer effects of anti-PD-1 antibody and 5-fluorouracil. However, it remains unclear whether MHY1485 treatment alters the effects of radiation on tumor cells. In this study, the radiosensitizing effects of MHY1485 were investigated using murine CT26 and LLC cell lines. We examined mTOR signaling, tumor cell growth, colony formation, apoptosis, senescence, oxidative stress, p21 accumulation and endoplasmic reticulum (ER) stress levels in cells treated with MHY1485 and radiation, either alone or together. We found that MHY1485 treatment inhibited growth and colony formation in both cell lines under irradiation and no-irradiation conditions, results that were not fully consistent with MHY1485's known role in activating mTOR signaling. Furthermore, we found that combined treatment with MHY1485 and radiation significantly increased apoptosis and senescence in tumor cells in association with oxidative stress, ER stress and p21 stabilization, compared to radiation treatment alone. Our results suggested that MHY1485 enhances the radiosensitivity of tumor cells by a mechanism that may differ from MHY1485's role in mTOR activation.
Assuntos
Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Morfolinas/farmacologia , Proteínas de Neoplasias/agonistas , Serina-Treonina Quinases TOR/efeitos dos fármacos , Triazinas/farmacologia , Animais , Apoptose/efeitos da radiação , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Senescência Celular/efeitos da radiação , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos da radiação , Genes p53 , Genes ras , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ensaio Tumoral de Célula-TroncoRESUMO
Malignant melanoma is a critical and aggressive skin tumor with a steeply rising incidence and a less favorable prognosis due to the lack of efficient treatment. Photodynamic therapy (PDT) is a new promising treatment for this tumor through photosensitizers-mediated oxidative cytotoxicity. In this study, we explored the role of berberine-mediated PDT (BBR-PDT) in the anti-proliferative effect on human malignant melanoma cells (MMCs). We found that there were significant differences between MMCs with BBR-PDT and MMCs with BBR or PDT only. Further research showed that BBR-PDT induced apoptosis via up-regulating the expression of cleaved caspase-3 protein. We also observed that LC3-related autophagy level was upregulated in MMCs with BBR-PDT. Besides, it was also found that BBR-PDT activated endoplasmic reticulum (ER) stress, involving a dramatic increase in reactive oxygen species (ROS). Interestingly, the knockdown of CHOP protein expression inhibited apoptosis, autophagy and ER stress levels caused by BBR-PDT, suggesting that CHOP protein may be related to apoptosis, autophagy and ER stress in MMCs with BBR-PDT. Collectively, our results indicated that BBR-PDT had an essential impact on MMCs' growth inhibition, and therefore may reveal the possibility of developing BBR-PDT into human malignant melanoma.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Berberina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melanoma/terapia , Fotoquimioterapia/métodos , Fator de Transcrição CHOP/metabolismo , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Berberina/química , Western Blotting , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos da radiação , Humanos , Luz , Melanoma/metabolismo , Melanoma/patologia , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
Autophagy has been reported to play a radioresistance role in high-dose-rate irradiation. However, its mechanisms and roles in continuous low-dose-rate (CLDR) irradiation have not been clearly understood. Iodine-125 (I-125) seed brachytherapy is a modality of CLDR irradiation and has been used in the treatment of various cancers. In this study, we investigated the mechanisms and roles of autophagy induced by I-125 seed radiation in human esophageal squamous cell carcinoma (ESCC) cell lines (Eca-109 and EC-109) and a xenograft mouse model. The results of this work showed that I-125 seed radiation induced a dose-dependent increase in autophagy in both cell lines. In Eca-109 cells, I-125 seed radiation-induced endoplasmic reticulum (ER) stress, manifesting as the increased levels of intracellular Ca2+ and Grp78/BiP, and activated PERK-eIF2α, IRE1, and ATF6 pathways of the unfolded protein response. Knockdown of PERK led to the decreased expression of autophagy marker, LC3B-II. Inhibition of autophagy by chloroquine or knockdown of ATG5 enhanced I-125 seed radiation-induced cell proliferation inhibition and apoptosis. Interestingly, chloroquine did not aggravate ER stress but promoted apoptosis via the mitochondrial pathway. The animal experiment showed that inhibition of autophagy by chloroquine improved the efficacy of I-125 seed radiation. In summary, our data demonstrate that I-125 seed CLDR radiation induces ER stress-mediated autophagy in ESCC. Autophagy plays a pro-survival role in I-125 seed CLDR irradiation, and chloroquine is a potential candidate for use in combination therapy with I-125 seed radiation treatment to improve efficacy against ESCC.
Assuntos
Autofagia/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos da radiação , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/radioterapia , Radioisótopos do Iodo/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Chaperona BiP do Retículo Endoplasmático , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiaçãoRESUMO
Excessive light exposure is a principal environmental factor, which can cause damage to photoreceptors and retinal pigment epithelium (RPE) cells and may accelerate the progression of age-related macular degeneration (AMD). In this study, oxidative stress, endoplasmic reticulum (ER) stress and autophagy caused by light exposure were evaluated in vitro and in vivo. Light exposure caused severe photo-oxidative stress and ER stress in photoreceptors (661W cells) and RPE cells (ARPE-19 cells). Suppressing either oxidative stress or ER stress was protective against light damage in 661W and ARPE-19 cells and N-acetyl-L-cysteine treatment markedly inhibited the activation of ER stress caused by light exposure. Moreover, suppressing autophagy with 3-methyladenine significantly attenuated light-induced cell death. Additionally, inhibiting ER stress either by knocking down PERK signals or with GSK2606414 treatment remarkably suppressed prolonged autophagy and protected the cells against light injury. In vivo experiments verified neuroprotection via inhibiting ER stress-related autophagy in light-damaged retinas of mice. In conclusion, the above results suggest that light-induced photo-oxidative stress may trigger subsequent activation of ER stress and prolonged autophagy in photoreceptors and RPE cells. Suppressing ER stress may abrogate over-activated autophagy and protect the retina against light injury.
Assuntos
Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Luz/efeitos adversos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Antioxidantes/farmacologia , Autofagia/efeitos da radiação , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos da radiação , Humanos , Indóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos da radiação , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/efeitos da radiação , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: Endoplasmic reticulum (ER) calcium depletion-induced ER stress is a crucial signal for keratinocyte differentiation and barrier homeostasis, but its effects on the epidermal tight junction (TJ) have not been characterized. Ultraviolet B (UVB) causes ER calcium release in keratinocytes and disrupts epidermal TJ, however, the involvement of ER stress in the UVB-induced TJ alterations remains unknown. OBJECTIVES: To investigate the effect of ER stress by pharmacological ER calcium depletion or UVB on the TJ integrity in normal human epidermal keratinocytes (NHEK). METHODS: NHEK were exposed to ER calcium pump inhibitor thapsigargin (Tg) or UVB. ER stress markers and TJ molecules expression, TJ and F-actin structures, and TJ barrier function were analyzed. RESULTS: Tg or UVB exposure dose-dependently triggered unfolded protein response (UPR) in NHEK. Low dose Tg induced the IRE1α-XBP1 pathway and strengthened TJ barrier. Contrary, high dose Tg activated PERK phosphorylation and disrupted TJ by F-actin disorganization. UVB disrupted TJ and F-actin structures dose dependently. IRE1α RNase inhibition induced or exacerbated TJ and F-actin disruption in the presence of low dose Tg or UVB. High dose Tg increased RhoA activity. 4-PBA or Rho kinase (ROCK) inhibitor partially prevented the disruption of TJ and F-actin following high dose Tg or UVB. CONCLUSIONS: ER stress has bimodal effects on the epidermal TJ depending on its intensity. The IRE1α pathway is critical for the maintenance of TJ integrity during mild ER stress. Severe ER stress-induced UPR or ROCK signalling mediates the disruption of TJ through cytoskeletal disorganization during severe ER stress.
Assuntos
Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos da radiação , Queratinócitos/patologia , Junções Íntimas/patologia , Raios Ultravioleta/efeitos adversos , Amidas/farmacologia , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Retículo Endoplasmático/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Fenilbutiratos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/efeitos da radiação , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos da radiação , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Immunogenic cell death (ICD) elicited by photodynamic therapy (PDT) is mediated through generation of reactive oxygen species (ROS) that induce endoplasmic reticulum (ER) stress. However, the half-life of ROS is very short and the intracellular diffusion depth is limited, which impairs ER localization and thus limits ER stress induction. To solve the problem, we synthesized reduction-sensitive Ds-sP NPs (PEG-s-s-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] nanoparticles) loaded with an efficient ER-targeting photosensitizer TCPP-TER (4,4',4â³,4'â³-(porphyrin-5,10,15,20-tetrayl)tetrakis(N-(2-((4-methylphenyl)sulfonamido)ethyl)benzamide). The resulting Ds-sP/TCPP-TER NPs could selectively accumulate in the ER and locally generate ROS under near-infrared (NIR) laser irradiation, which induced ER stress, amplified ICD, and activated immune cells, leading to augmented immunotherapy effect. This study presents a novel ICD amplifying, ER-targeting PDT strategy that can effectively eradicate primary tumors under NIR exposure, as well as distant tumors through an abscopal effect.
Assuntos
Sistemas de Liberação de Medicamentos , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Imunoterapia , Raios Infravermelhos , Neoplasias Experimentais , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Linhagem Celular Tumoral , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/imunologia , Estresse do Retículo Endoplasmático/efeitos da radiação , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Espécies Reativas de Oxigênio/imunologiaRESUMO
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced in response to endoplasmic reticulum (ER) stress induced by various stressors, but its involvement in the radioresistance of tumor cells is unknown. Here, we found that MANF is released after γ-irradiation (2 Gy and 4 Gy) of B16 melanoma cells, and its release was suppressed by 4-phenylbutyric acid, an ER stress inhibitor. MANF was not released after low-dose (1 Gy) γ-irradiation, but pretreatment of 1 Gy-irradiated cells with recombinant MANF enhanced the cellular DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the DNA damage response and p53 activation by γ-irradiation (2 Gy) were suppressed, and reproductive cell death was increased. MANF also activated the ERK signaling pathway. Our findings raise the possibility that MANF could be a new target for overcoming radioresistance.
Assuntos
Estresse do Retículo Endoplasmático/efeitos da radiação , Retículo Endoplasmático/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Fatores de Crescimento Neural/genética , Tolerância a Radiação/genética , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Raios gama , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/radioterapia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/metabolismo , Fenilbutiratos/farmacologia , Fosforilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Endoplasmic reticulum (ER) stress is a conserved cellular process for cells to clear unfolded or misfolded proteins and maintain cell homeostasis under stress conditions. Autophagy may act as a pro-survival strategy to cope with multiple stress conditions in tumor progression and distant metastasis. Although many studies have demonstrated that there is a close correlation between radiation-induced ER stress and autophagy, the molecular mechanisms currently remain unclear. In the present study, we performed an in vivo study concerning the effect of autophagy induced by ER stress on the radiosensitivity of mouse sarcoma using X-rays. Our results documented that X-rays could induce ER stress in sarcoma and then autophagy was activated by unfolded protein response (UPR) through the IRE1-JNK-pBcl2-Beclin1 signaling axis. The induction of autophagy caused a decline in cell apoptosis while inhibiting the autophagy resulted in increased apoptosis and inhibition of tumor progression. Combined treatment of X-ray exposure and chloroquine increased ER stress-related apoptosis and enhanced the radiosensitivity of mouse sarcoma that was not sensitive to X-ray irradiation alone. Thus, our study indicates that inhibition of ER stress-induced autophagy might be a novel strategy to improve the efficacy of radiotherapy against radioresistant sarcoma.
Assuntos
Antimaláricos/uso terapêutico , Apoptose/efeitos da radiação , Autofagia/efeitos da radiação , Cloroquina/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos da radiação , Sarcoma/radioterapia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Tolerância a Radiação/efeitos dos fármacosRESUMO
It has been widely reported that ultraviolet-B (UV-B) radiation is the main extrinsic etiological agent that causes skin photodamage. UV-B exposure mediated photodamage (photo-aging/photo-carcinogenesis) to human skin is caused due to several physiological events at tissue, cellular and molecular levels that lead to impairment of skin function and integrity. In the present study, we investigated the protective role of Trigonelline (TG) against UV-B induced photo-damage in Human Dermal Fibroblasts (Hs68 cells) and Balb/C mice. We exposed human skin fibroblasts and Balb/C mice to UV-B radiation and evaluated various parameters of cellular damage, including, oxidative stress, cytosolic calcium (Ca2+) levels, apoptotic and ER-stress marker proteins. We found that UV-B irradiation induced ROS generation lead to the depletion of endoplasmic reticulum (ER) calcium and increased the expression of ER stress protein markers (phosphorylated elf2α, CHOP, ATF4) as well as apoptotic protein markers (Bcl2, Bax and caspase-9) in a dose and time dependent manner in Hs68 cells. We then determined the effect of TG treatment on UV-B -induced cell death in Hs68 cells and observed that cells exposed to UV-B radiation and treated with TG had a significantly higher survival rate compared to cells exposed to UV-B radiation alone. TG treatment successfully reduced oxidative stress; restored Ca2+ homeostasis and re-established the ER function and prevented apoptotic cell death process. Our results suggest that TG can be used as a potential therapeutic/cosmeceutic agent in preventing skin photo-damage.
Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Raios Ultravioleta , Animais , Apoptose/efeitos da radiação , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos da radiação , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
Resveratrol displays cardioprotective activity; however, its mechanism of action remains unclear. In the current study, resveratrol-induced myocardial protection from endoplasmic reticulum stress (ERS) was investigated, focusing on the roles of Zn2+ and the mitochondrial permeability transition pore (mPTP). We found, using the MTT/LDH kit, that 2-DG-induced ERS significantly decreased H9c2 cell viability. Resveratrol markedly inhibited the expression of endoplasmic reticulum chaperone GRP 78/94 and ERS-related apoptosis proteins CHOP, Caspase12, and JNK induced by 2-DG. The zinc ion chelator TPEN, and ERK/GSK-3ß inhibitors PD98059 and SB216763 and their siRNAs blocked resveratrol function. The AKT inhibitor LY294002 and siRNA did not alter the action of resveratrol. In addition, resveratrol significantly increased the phosphorylation of ERK and GSK-3ß. Resveratrol prevented 2-DG-induced mPTP opening and increased intracellular Zn2+ concentration indicated by TMRE and Newport Green DCF fluorescence intensity, which were further abrogated by ERK/GSK-3ß inhibitors and siRNAs. Our data suggested that resveratrol protected cardiac cells from ERS by mobilizing intracellular Zn2+ and preventing mPTP opening through the ERK/GSK-3ß but not PI3K/AKT signaling pathway.
Assuntos
Cardiotônicos/farmacologia , Estresse do Retículo Endoplasmático/efeitos da radiação , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Resveratrol/farmacologia , Zinco/metabolismo , Animais , Linhagem Celular , Cromonas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Morfolinas/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Transdução de SinaisRESUMO
DNA damage associated with assisted reproductive technologies is an important factor affecting gamete fertility and embryo development. Activation of the TGR5 receptor by tauroursodeoxycholic acid (TUDCA) has been shown to reduce endoplasmic reticulum (ER) stress in embryos; however, its effect on genome damage responses (GDR) activation to facilitate DNA damage repair has not been examined. This study aimed to investigate the effect of TUDCA on DNA damage repair and embryo development. In a porcine model of ultraviolet light (UV)-induced nuclear stress, TUDCA reduced DNA damage and ER stress in developing embryos, as measured by γH2AX and glucose-regulated protein 78 immunofluorescence, respectively. TUDCA was equally able to rescue early embryo development. No difference in total cell number, DNA damage, or percentage of apoptotic cells, measured by cleaved caspase 3 immunofluorescence, was noted in embryos that reached the blastocyst stage. Interestingly, Dicer-substrate short interfering RNA-mediated disruption of TGR5 signaling abrogated the beneficial effects of TUDCA on UV-treated embryos. Quantitative PCR analysis revealed activation of the GDR, through increased messenger RNA abundance of DNAPK, 53BP1, and DNA ligase IV, as well as the ER stress response, through increased spliced XBP1 and X-linked inhibitor of apoptosis. Results from this study demonstrated that TUDCA activates TGR5-mediated signaling to reduce DNA damage and improve embryo development after UV exposure.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Suínos/embriologia , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Blastocisto/citologia , Blastocisto/efeitos da radiação , Células Cultivadas , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/efeitos da radiação , Feminino , Fertilização In Vitro/métodos , Técnicas de Silenciamento de Genes , Técnicas de Maturação in Vitro de Oócitos/métodos , Recuperação de Oócitos/métodos , Ovário/citologia , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Raios Ultravioleta , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/efeitos da radiação , Zigoto/efeitos da radiaçãoRESUMO
BACKGROUND: Retinal pigment epithelium cells (RPEs) are critical for maintaining retinal homeostasis. Accumulation of age-related lipofuscin, N-retinylidene-N-retinylethanolamine (A2E), makes RPEs vulnerable to blue light-mediated damage, which represents an initial cause of some retinal degenerative diseases. This study investigated the activation of autophagy and the signaling pathway involved in glucose-related protein 78 (GRP78) induced autophagy in blue light-mediated damage of A2E-laden RPEs. In addition, we explored whether autophagy could play a protective role by alleviating endoplasmic reticulum (ER) stress to promote RPEs survival. METHODS: RPEs were incubated with 25 µM A2E for 2 h and exposed to blue light for 20 min. The expression of ER stress-related apoptotic proteins, CHOP and caspase-12, as well as autophagy marker LC3 were measured by western blot analysis. Autophagosomes were observed by both transmission electron microscopy and immunofluorescence assays. GRP78 interference performed by short hairpin RNA (shRNA) was used to identify the signaling pathway involved in GRP78 induced autophagy. Cell death was assessed using TUNEL analysis. RESULTS: Treatment with A2E and blue light markedly increased the expression of ER stress-related apoptotic molecules CHOP and caspase-12. The activation of autophagy was recognized by observing autophagosomes at ultrastructural level. Additionally, punctate distributions of LC3 immunofluorescence and enhanced conversions of LC3-I to LC3-II were found in A2E and blue light-treated RPEs. Moreover, GRP78 interference reduced AMPK phosphorylation and promoted mTOR activity, thereby downregulating autophagy. In addition, the inhibition of autophagy made RPEs vulnerable to A2E and blue light damage. In contrast, the autophagy inducer rapamycin alleviated ER stress to promote RPEs survival. CONCLUSIONS: GRP78 activates autophagy via AMPK/mTOR in blue light-mediated damage of A2E-laden RPEs in vitro. Autophagy may be a vital endogenous cytoprotective process to alleviate stress for RPEs survival in retinal degenerative diseases.
Assuntos
Autofagia/fisiologia , Estresse do Retículo Endoplasmático , Células Epiteliais , Proteínas de Choque Térmico/farmacologia , Epitélio Pigmentado da Retina , Retinoides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos da radiação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/efeitos da radiação , Humanos , Luz , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos da radiação , Transdução de Sinais/fisiologiaRESUMO
C/EBP-ß, a basic leucine zipper transcription factor, has important roles in the regulation of the body immune and inflammatory responses. Wistar rats subjected to combined irradiation were characterized by an increase in the content of the C/EBP-ß LIP isoform in the pituitary gland. The obtained data indicate that moderate doses of ionizing radiation to initiate the endoplasmic reticulum stress response and are likely to initiate C/EBP-ß-mediated cell death according to the apoptotic scenario. This study also confirms the earlier hypothesis about the alterations of the hypothalamic-pituitary-adrenocortical axis in response to moderate doses of ionizing radiation.
Assuntos
Apoptose/efeitos da radiação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Radioisótopos de Carbono , Estresse do Retículo Endoplasmático/efeitos da radiação , Raios gama/efeitos adversos , Hipófise/metabolismo , Animais , Hipófise/patologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
Overcoming resistance to radiation is a great challenge in cancer therapy. Here, we highlight that targeting valosin-containing protein (VCP) improves radiation sensitivity in esophageal squamous cell carcinoma (ESCC) cell lines and show the potential of using VCP as a prognosis marker in locally advanced ESCC treated with radiation therapy. Esophageal squamous cell carcinoma cell lines with high VCP expression were treated with VCP inhibitor combined with radiotherapy. Cell proliferation, colony formation, cell death, and endoplasmic reticulum (ER) stress signaling were evaluated. Moreover, patients with newly diagnosed locally advanced ESCC who were treated with radiotherapy were analyzed. Immunohistochemistry was used to detect the expression of VCP. The correlation between overall survival and VCP was investigated. Esophageal squamous cell carcinoma cells treated with VCP inhibitor and radiotherapy showed attenuated cell proliferation and colony formation and enhanced apoptosis. Further investigation showed this combined strategy activated the ER stress signaling involved in unfolded protein response, and inhibited the ER-associated degradation (ERAD) pathway. Clinical analysis revealed a significant survival benefit in the low VCP expression group. Targeting VCP resulted in antitumor activity and enhanced the efficacy of radiation therapy in ESCC cells in vitro. Valosin-containing protein is a promising and novel target. In patients with locally advanced ESCC who received radiotherapy, VCP can be considered as a useful prognostic indicator of overall survival. Valosin-containing protein inhibitors could be developed for use as effective cancer therapies, in combination with radiation therapy.
Assuntos
Quimiorradioterapia/métodos , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas do Esôfago/terapia , Proteínas de Neoplasias/antagonistas & inibidores , Tolerância a Radiação , Proteína com Valosina/antagonistas & inibidores , Acetanilidas/farmacologia , Idoso , Análise de Variância , Protocolos de Quimioterapia Combinada Antineoplásica , Benzotiazóis/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Terapia Combinada/métodos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos da radiação , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/mortalidade , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Modelos de Riscos Proporcionais , Radioterapia Conformacional , Ensaio Tumoral de Célula-Tronco , Proteína com Valosina/metabolismoRESUMO
Aberrant increase in angiogenesis contributes to the progression of malignant solid tumors. An alternative antiangiogenesis therapy is critical for cancer, since the current antiangiogenesis drugs lack specificity for tumor cells. In the present study, the effects and mechanisms of lowintensity pulsed ultrasound (LIPUS) on human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMECs) were investigated, and the therapeutic potential of this technology was assessed. HUVECs and HMECs were treated with LIPUS (0.5 MHz; 210 mW/cm2) for 1 min and cultured for 24 h. Flow cytometry and Cell Counting Kit8 assays demonstrated that LIPUS treatment at a dose of 210 mW/cm2 promoted apoptosis and decreased the viability in HUVECs and HMECs. Realtime cell analysis also revealed that LIPUS did not affect the proliferation or migration of HUVECs. An endothelial cell tube formation assay indicated that LIPUS treatment inhibited the angiogenic ability of HUVECs and HMECs. Furthermore, LIPUS increased the protein levels of the apoptosisassociated cleaved Caspase3 and decreased the Bcell lymphoma2 levels. LIPUS increased the phosphorylation of p38 mitogenactivated protein kinase (MAPK), and the levels of endoplasmic reticulum (ER) stressassociated markers, including activating transcription factor4 (ATF4) and phosphorylated eukaryotic initiation factor 2α (eIF2α). The p38 inhibitor SB203580 reversed the proapoptotic and antiangiogenic effects of LIPUS in cells. Finally, inhibition of p38 decreased the LIPUSinduced elevation of peIF2α and ATF4 levels. Taken together, these results suggested that LIPUS promoted apoptosis and inhibited angiogenesis in human endothelial cells via the activation of p38 MAPKmediated ER stress signaling.
Assuntos
Apoptose/efeitos da radiação , Estresse do Retículo Endoplasmático/efeitos da radiação , Células Endoteliais/efeitos da radiação , Neovascularização Patológica/radioterapia , Ondas Ultrassônicas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Proliferação de Células/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Linfoma de Células B/radioterapia , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS: In this study, we examined whether ß-apopicropodophyllin (APP) could act as a radiosensitizer in non-small cell lung cancer (NSCLC) cells. MAIN METHODS: The in vitro radiosensitizing activity of APP was demonstrated with clonogenic assay, immunoblotting, Annexin V-Propidium iodide (PI) assay, BrdU incorporation, detection of mitochondrial ROS/intracellular of H2O2, mitochondrial membrane potential detection, and performing of isolation of mitochondrial and cytosolic fractions. The in vivo radiosensitizing activity of APP was determined in xenografted mice with co-treatment of APP and IR based on measurement of tumor volumes and apoptotic cell death. KEY FINDINGS: The results of a clonogenic assay indicated that a combination of APP and γ-ionizing radiation (IR) inhibits cell growth and increases cell death in NSCLC cells. Several signal transduction pathways were examined for their potential involvement in the apparent radiosensitization effect of APP, as assessed by immunoblotting analyses and mitochondrial potential determination in vitro. Treatment of NCI-H460 cells with 15 nM APP and NCI-H1299 cells with 10 nM APP yielded dose-enhancement ratios of 1.44 and 1.24, respectively. Enhanced ER stress, disrupted mitochondrial membrane potential, and increased reactive oxygen species (ROS) were observed in cells co-treated with APP and IR, and this was followed by the cytosolic release of cytochrome c and consequent activation of caspase-3 and -9. Notably, inhibition of JNK, which prevents caspase activation, blocked the APP/IR-induced activations of ER stress and apoptotic cell death. In NCI-H460 or NCI-H1299 cell-xenografted mice, APP/IR treatment delayed the time it took tumors to reach a threshold size by 22.38 and 16.83 days, respectively, compared with controls, to yield enhancement factors of 1.53 and 1.38, respectively. SIGNIFICANCE: APP has a radiosensitizing function derived from its ability to induce apoptotic cell death via activation of ER stress, disruption of mitochondrial membrane potential, and induction of the caspase pathway.